PD-L1 Positivity Research Articles

Development of a Syrian hamster anti-PD-L1 monoclonal antibody enables oncolytic adenoviral immunotherapy modelling in an immunocompetent virus replication permissive setting.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer, but preclinical testing of hypotheses such as combination therapies has been complicated, in part due to species incompatibility issues. For example, one of few known permissive animal models for oncolytic adenoviruses is the Syrian hamster, for which an ICI, mainly an anti-PD-L1 monoclonal antibody (mAb) was not previously available. In this study, we developed an anti-Syrian hamster PD-L1 mAb to enable the evaluation of safety and efficacy, when combining anti-PD-L1 with an oncolytic adenovirus encoding tumour necrosis factor alpha (TNFα) and interleukin-2 (IL-2) (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2 or TILT-123). Recombinant Syrian hamster PD-L1 was expressed and mice immunized for mAb formation using hybridoma technology. Clonal selection through binding and functional studies in vitro, in silico and in vivo identified anti-PD-L1 clone 11B12-1 as the primary mAb candidate for immunotherapy modelling. The oncolytic virus (OV) and ICI combination approach was then evaluated using 11B12-1 and TILT-123 in a Syrian hamster model of pancreatic ductal adenocarcinoma (PDAC). Supernatants from hybridoma parent subclone 11B12B4 provided the highest positive PD-L1 signal, on Syrian hamster PBMCs and three cancer cell lines (HT100, HapT1 and HCPC1). In vitro co-cultures revealed superior immune modulated profiles of cell line matched HT100 tumour infiltrating lymphocytes when using subclones of 7G2, 11B12 and 12F1. Epitope binning and epitope prediction using AlphaFold2 and ColabFold revealed two distinct functional epitopes for clone 11B12-1 and 12F1-1. Treatment of Syrian hamsters bearing HapT1 tumours, with 11B12-1 induced significantly better (p<0.05) tumour growth control than isotype control by day 12. 12F1-1 did not induce significant tumour growth control. The combination of 11B12-1 with oncolytic adenovirus TILT-123 improved tumour growth control further, when compared to monotherapy (p<0.05) by day 26. Novel Syrian hamster anti-PD-L1 clone 11B12-1 induces tumour growth control in a hamster model of PDAC. Combining 11B12-1 with oncolytic adenovirus TILT-123 improves tumour growth control further and demonstrates good safety and toxicity profiles.

Characterisation of tumour-immune phenotypes and PD-L1 positivity in squamous bladder cancer

AimsImmune checkpoint inhibitor (ICI) therapy has become a viable treatment strategy in bladder cancer. However, treatment responses vary, and improved biomarkers are needed. Crucially, the characteristics of immune cells remain understudied especially in squamous differentiated bladder cancer (sq-BLCA). Here, we quantitatively analysed the tumour-immune phenotypes of sq-BLCA and correlated them with PD-L1 expression and FGFR3 mutation status.Methods Tissue microarrays (TMA) of n = 68 non-schistosomiasis associated pure squamous cell carcinoma (SCC) and n = 46 mixed urothelial carcinoma with squamous differentiation (MIX) were subjected to immunohistochemistry for CD3, CD4, CD8, CD56, CD68, CD79A, CD163, Ki67, perforin and chloroacetate esterase staining. Quantitative image evaluation was performed via digital image analysis.ResultsImmune infiltration was generally higher in stroma than in tumour regions. B-cells (CD79A) were almost exclusively found in stromal areas (sTILs), T-lymphocytes and macrophages were also present in tumour cell areas (iTILs), while natural killer cells (CD56) were nearly missing in any area. Tumour-immune phenotype distribution differed depending on the immune cell subset, however, hot tumour-immune phenotypes (high density of immune cells in tumour areas) were frequently found for CD8 + T-cells (33%), especially perforin + lymphocytes (52.2%), and CD68 + macrophages (37.6%). Perforin + CD8 lymphocytes predicted improved overall survival in sq-BLCA while high PD-L1 expression (CPS ≥ 10) was significantly associated with higher CD3 + , CD8 + and CD163 + immune cell density and high Ki67 (density) of tumour cells. Furthermore, PD-L1 expression was positively associated with CD3 + /CD4 + , CD3 + /CD8 + and CD68 + /CD163 + hot tumour-immune phenotypes. FGFR3 mutation status was inversely associated with CD8 + , perforin + and CD79A + lymphocyte density.ConclusionsComputer-based image analysis is an efficient tool to analyse immune topographies in squamous bladder cancer. Hot tumour-immune phenotypes with strong PD-L1 expression might pose a promising subgroup for clinically successful ICI therapy in squamous bladder cancer and warrant further investigation.

Subtyping of advanced lung cancer based on PD-L1 expression, tumor histopathology and mutation burden (EGFR and KRAS): a study from North India.

Immune checkpoint inhibitor (PD-L1) therapy of advanced non-small-cell lung cancer (NSCLC) has variable outcomes. Tumor subtypes based on PD-L1 expression, histopathology, mutation burden is required for patient stratification and formulation of treatment guidelines. Lung cancers (n=57) diagnosed at Pathology department, VPCI (2018-2021) were retrospectively analyzed. PD-L1(SP263) expressed by tumor cells [low (<1%), medium (1-49%), high (≥50%)] was correlated with histopathology, microenvironment, EGFR, KRAS expression. Patients were categorized into high and low risk based on their: i) gender: males (n=47, 30-89 years), females (n=10, 45-80 years); ii) smoking history: males 26/47 (45.61%), females 1/10 (10%); iii) tumor subtyping: squamous cell carcinoma 15/57 (26.32%), adenocarcinoma 6/57 (17.54%), NSCLC-undifferentiated 24/57 (42.10%), adenosquamous carcinoma 5/57 (8.77 %), carcinosarcoma 4/57 (7.02%), small cell carcinoma 1/57 (1.75%); iv) inflammatory tumor microenvironment/TILs 44/57 (77.1%); iv) PD-L1 positivity-31/57 (54.3%); v) concomitant EGFR/KRAS positivity. PD-L1positive cases showed squamous/undifferentiated histopathology, concomitant EGFR+ (9/20, 45%) and KRAS+ (8/15, 53.3%), smoking+ (21/31,67.74%).PD-L1 negative cases (26/57, 45.6%), were EGFR+ (2/14, 14.28%) and KRAS+ (6/19, 31.5%). The high-risk lung cancer subtypes show squamous/undifferentiated histopathology, inflammatory microenvironment, male preponderance, smoking history, higher concomitant PD-L1, KRAS and EGFR positivity. Lung cancer subtyping can predict clinical response/resistance of patients prior to initiation of PD-L1 inhibitor therapies and can be used to guide therapy.

Intertumor heterogeneity of locally advanced gastric cancer.

435 Background: The aim of the present study was to determine to the effect of HER2, dMMR, PD-L1 status in the primary tumor and in metastasis to the regional lymph node of GC patients. Methods: A total of 44 patients with primary loco-regional (N+) gastric adenocarcinoma were recruited, and two formalin-fixed paraffin-embedded (FFPE) tumor-containing blocks of each patient (one block with primary tumor and one block with metastasis to the regional lymph node)were selected for HER2, dMMR, PD-L1 (CPS) immunohistochemical (IHC) assay. Clinicopathological characteristics were recorded, and intertumoral heterogeneity of HER2, dMMR, PD-L1 IHC expression was determined. Results: The study included 19 women and 25 men, 30 patients had Laurén’s intestinal type of cancer, 14 had a Laurén’s diffuse type. In 5 patients (11.4%) overexpression of Her-2 neu was detected, and heterogeneity was detected in all five cases: in three patients with HER2 overexpression in the primary tumor, no overexpression was detected in the metastasis to the regional lymph node, in two cases overexpression was detected in the metastasis, while no overexpression was observed in the primary tumor. 2 patients showed signs of dMMR (4.5%), in one case, heterogeneity in dMMR status was also revealed: signs of dMMR in the primary tumor and its absence in regional metastasis (pMMR). Assessment of PD-L1 status was carried out using the CPS formula. Ten patients (22.7%) had a negative PD-L1 status (CPS=0) both in the primary tumor and in regional metastasis. Positive PD-L1 status in the primary tumor: in 24 patients CPS= 0-5 (54.5%), in 5 patients CPS= 5-10 (11.4%) and 5 patients CPS= 10 or more (11.4% ). Positive PD-L1 status in regional lymph node metastasis: in 24 patients CPS= 0-5 (54.5%), in 5 patients CPS= 5-10 (11.4%) and in 5 patients CPS= 10 or more ( 11.4%). In 5 cases (11.4%) marked heterogeneity in PD-L1 status between the primary tumor and its regional metastasis (1 case with dMMR heterogeneity). Conclusions: The research findings in this paper indicate that the intertumoral heterogeneity of HER2 overexpression is common in GC patients, We highly recommend multi-block HER2 assessment for accurate diagnosis of GC. Given the rarity of status detection dMMR, a study in a larger number of patients is required, however, our data also confirm the presence of heterogeneity in dMMR status, and probably the possibility of metastasis of a more aggressive pMMR clone. Heterogeneity in PD-L1 status is quite rare and amounted to only 11.4%, which also has important practical significance.

Targeting the Tumor Immune Microenvironment Could Become a Potential Therapeutic Modality for Aggressive Pituitary Adenoma

This study aimed to explore the relationship between the aggressiveness and immune cell infiltration in pituitary adenoma (PA) and to provide the basis for immuno-targeting therapies. One hundred and three patients with PA who underwent surgery at a single institution were retrospectively identified. The infiltration of macrophages and T-lymphocytes was quantitatively assessed. The number of CD68+ macrophages was positively correlated with Knosp (p = 0.003) and MMP-9 expression grades (p = 0.00). The infiltration of CD163+ macrophages differed among Knosp (p = 0.022) and MMP-9 grades (p = 0.04). CD8+ tumor-infiltrating lymphocytes (TILs) were also positively associated with Knosp (p = 0.002) and MMP-9 grades (p = 0.01). Interestingly, MGMT expression was positively correlated with MMP-9 staining extent (p = 0.000). The quantities of CD8+ TILs (p = 0.016), CD68+ macrophages (p = 0.000), and CD163+ macrophages (p = 0.043) were negatively associated with MGMT expression levels. The number of CD68+ macrophages in the PD-L1 negative group was significantly more than that in the PD-L1 positive group (p = 0.01). The rate of PD-L1 positivity was positively correlated with the Ki-67 index (p = 0.046) and p53 expression (p = 0.029). Targeted therapy for macrophages and CD8+ TILs could be a helpful treatment in the future for aggressive PA. Anti-PD-L1 therapy may better respond to PAs with higher Ki-67 and p53 expression and more infiltrating CD68+ macrophages. Multiple treatment modalities, especially combined with immunotherapy could become a novel therapeutic strategy for aggressive PA.

Early Triple-Negative Breast Cancers in a Singapore Cohort Exhibit High PIK3CA Mutation Rates Associated With Low PD-L1 Expression

Mutations in the PI3K pathway, particularly PIK3CA, were reported to be intimately associated with triple-negative breast cancer (TNBC) progression and the development of treatment resistance. We profiled PIK3CA and other genes on 166 early-stage TNBC tumors from Singapore for comparison to publicly available TNBC cohorts. These tumors were profiled transcriptionally using a NanoString panel of immune genes and multiplex immunohistochemistry, then manually scored for PD-L1-positivity using 2 clinically relevant clones, SP142 and 22C3. We discovered a higher rate of PIK3CA mutations in our TNBC cohort than in non-Asian cohorts, along with TP53, BRCA1, PTPN11, and MAP3K1 alterations. PIK3CA mutations did not affect overall or recurrence-free survival, and when compared with PIK3CAWT tumors, there were no differences in immune infiltration. Using 2 clinically approved antibodies, PIK3CAmut tumors were associated with PD-L1 negativity. Analysis of comutation frequencies further revealed that PIK3CA mutations tended to be accompanied by MAP kinase pathway mutation. The mechanism and impact of PIK3CA alterations on the TNBC tumor immune microenvironment and PD-L1 positivity warrant further study.

Estrogen receptor and programmed death ligand-1 expression in type 1 endometrial cancer and its associated clinicopathological characteristics

BackgroundThis study aimed to determine the association of estrogen receptor (ER) and programmed death ligand-1 (PD-L1) expression with the clinicopathological characteristics of type 1 endometrial cancer. Materials and methodsA total of 85 patients with type 1 endometrial cancer who underwent surgery at the Dr. Soetomo Hospital, Surabaya, Indonesia were retrospectively studied. Data about the age, menopausal status, body mass index, disease stage, cell differentiation, angiolymphatic invasion, myometrial invasion, and adjuvant therapy of the patients were collected from medical records. Immunohistochemistry with ER and PD-L1 antibodies was performed on all samples. The association between ER and PD-L1 expression and clinicopathological characteristics was statistically analyzed. ResultsThe positivity rates of ER and PD-L1 in type 1 endometrial cancer were 68.2 % and 78.5 %, respectively. ER positivity was significantly correlated with body mass index (BMI) ≥25, premenopausal status, early stage of disease, <1/2 myometrial invasion, negative nodal metastasis, and lack of adjuvant therapy. It was also associated with age <55 years, low-grade cells, and angiolymphatic invasion, but the correlation was not significant. Meanwhile, PD-L1 positivity was significantly correlated with BMI <25, menopausal status, advanced stage of disease, high-grade cells, angiolymphatic invasion, and adjuvant therapy. It was also associated with age ≥55 years and nodal metastasis, but the correlation was not significant. ConclusionER and PDL-1 positivity is associated with the clinicopathological characteristics of type 1 endometrial cancer.

Clinicopathologic Features and Prognostic Significance of Immunohistochemistry and In Situ Hybridization Based Molecular Classification in Gastric Carcinoma.

Gastric carcinoma (GC) is a highly heterogeneous disease with many subtypes that have different morphologic and molecular characteristics. In the current study, we analyzed immunohistochemical (IHC) and in situ hybridization (ISH) features of GCs and evaluated their association with prognosis and clinicopathological features. Three hundred cases analyzed by IHC and ISH for microsatellite stability, p53, e-cadherin, HER2, PD-L1 expression, and Epstein-Barr virus (EBV) status. Cases were classified into five subgroups based on expression profile. The relationships between subgroups, clinicopathological features, and survival were determined. Ten (3.3%) cases were classified as EBV-associated, 45 (15%) as microsatellite instable (MSI), 73 (24.3%) as EBV-/microsatellite-stable (MSS)/epithelial-mesenchymal-transformation (EMT)-like, 75 (25%) as EBV-/MSS/ non-EMT-like/p53+, and 97 (32.3%) as EBV-/MSS/non-EMT-like/p53-. The MSI subtype had the best overall survival (OS). In contrast, the EBV-/MSS/EMT-like subtype had the poorest OS. The MSI subtype was also related with old age of the patient and antrum-corpus localized tumors, whereas the EBV-/MSS/EMT-like was associated with young age, larger tumor size, and advanced stage presentation. PD-L1 positivity is highly correlated with MSI and EBV-associated subtypes. Our data demonstrated a link between IHC/ISH characteristics of GC and clinical outcomes. IHC/ISH based molecular classification may be helpful in predicting the survival.

PD-L1 expression-related PI3K pathway correlates with immunotherapy efficacy in gastric cancer.

The programed death ligand-1 combined positive score (PD-L1 CPS), the only FDA-approved biomarker for immune checkpoint inhibitor therapy in gastric cancer (GC) patients, is an important but imperfect predictive biomarker. The molecular characteristics of tumors that influence the PD-L1 CPS are largely unknown and would be helpful for screening patients who would benefit from immunotherapy. PD-L1 immunohistochemistry (IHC) and targeted next-generation sequencing techniques were used to compare genomic alterations in 492 GC patients in two groups (PD-L1 CPS ⩾ 1, positive; CPS < 1, negative). Screened PD-L1 expression-related factors were analyzed for immunotherapy efficacy in three distinct GC cohorts from public databases. Positive PD-L1 expression occurred in 40% of GC patients and was associated with a higher proportion of phosphatidylinositol 3-kinase (PI3K), SWItch/Sucrose NonFermentable (SWI/SNF), lysine demethylase (KDM), and DNA (cytosine-5)-methyltransferase (DNMT) (all p < 0.01), pathway alterations. Compared to wild-type GC patients, those with PI3K pathway alterations had a higher response rate (p = 0.002) and durable clinical benefit rate with immunotherapy (p = 0.023, p = 0.038) as well as longer progression-free survival (p = 0.084, p = 0.0076) and overall survival (p = 0.2, p = 0.037) with immunotherapy. This study revealed PD-L1 expression-related factors in the tumor genome in a GC cohort. Alterations in the PI3K pathway associated with PD-L1 positivity were shown to be associated with better immunotherapy efficacy in three distinct GC cohorts from public databases. Our results provide a potential avenue for patient selection and rational immune combination development for GC patients.

Prognostic Significance of Immune-checkpoint Molecule PD-L1 in Classical Hodgkin Lymphoma: A Clinicopathologic Study of 120 Cases.

Classic Hodgkin lymphoma (cHL) is a common B-cell malignancy. Despite the good prognosis, in some patients the standard chemotherapy and radiotherapy-based approach does not lead to long-term remission, and these patients eventually relapse. Moreover, the primary refractory disease is of major concern regarding prognosis. We performed a retrospective analysis to evaluate PD-L1 expression in 120 patients with classic Hodgkin lymphoma (cHL). The median follow-up of the entire group of patients was 90 months. After initial therapy, complete remission was achieved in 113 (94.2%) patients. During the follow-up, cHL relapse/refractory disease was reported in 23 (19.2%) cases. A total of five patients died during the follow-up period, all from cHL progression. When determining PD-L1 expression on Hodgkin-Reed-Sternberg (HRS) cells, 37 cases (30.8%) were evaluated as negative, and 83 cases (69.2%) as positive. In the negative PD-L1 group of patients, no cHL relapse/refractory disease was observed during the follow-up period. However, out of 83 patients with positive PD-L1 expression on HRS cells, 23 (28%) showed relapse/refractory cHL. A significantly higher relapse rate was observed in PD-L1-positive patients diagnosed with cHL.

Predictive markers of immunotherapy in cervical cancer

Study of PD-L1 expression in squamous and adenosquamous cell cervical cancer (CC) by immunohistochemical (IHC) method, assessment of the relationship between PD-L1 tumor status and its clinical and morphological characteristics, TILs, MSI/dMMR, and HPV tumor status. Surgical material was obtained from 41 patients with CC, on which the expression of PD-L1, proteins of the MMR system and p16 was studied by the IHC method, the TILs index was determined. Positive PD-L1 status was found in 51.2% of the studied CC samples. In the study sample, the level of PD-L1 expression depended on the severity of lymphoid infiltration of the tumor (p=0.038), it was shown that a positive PD-L1 status of CC can be expected with a TILs value greater than or equal to 50%. The age of the patients, the histological variant of the tumor, the pT and pN stage, the presence of lymphovascular invasion, and the HPV status did not statistically significantly affect the level of PD-L1 expression, however, there was an association between the PD-L1 status and the grade of CC malignancy (p=0.027). The presence of the MSI/dMMR phenomenon was detected in a small percentage of carcinomas (4.9%), the PD-L1 status of these tumors was determined as positive. A positive PD-L1 status is determined in a significant number of cases of CC, regardless of most of the studied clinical and morphological characteristics; there is a statistically significant relationship between PD-L1 expression and the degree of tumor differentiation and TILs. It has been shown that CC with the MSI/dMMR phenomenon is characterized by a positive PD-L1 status. The authors consider it necessary to study the expression of PD-L1 in patients with cervical carcinomas in order to determine the possibility of prescribing personalized therapy with immune checkpoint inhibitors.

A retrospective analysis of tumor infiltrating lymphocytes in head and neck squamous cell carcinoma patients treated with nivolumab

Nivolumab, an immune checkpoint inhibitor is the first-line therapy for platinum-resistant recurrent/metastatic head and neck cancer, and highly effective for some patients. However, no factors have been identified that could predict response or prognosis after nivolumab administration. We retrospectively investigated the association between tumor infiltrating lymphocytes (TILs) of initial pathology and prognosis in patients treated with nivolumab. Twenty-eight patients with human papilloma virus and Epstein–Barr virus unrelated head and neck squamous cell carcinoma were enrolled. CD8+cells, FoxP3+cells and FoxP3−CD4+cells in the tumoral and peritumoral stromal area and PD-L1 were measured. In result, FoxP3−CD4+TIL, FoxP3+TIL, and CD8+TIL were not correlated with survival in either intratumoral and stromal area. In univariate analysis, objective response was significant prognostic factor both in progression-free survival and overall survival (p = 0.01, 0.006, respectively). PD-L1 was also significant prognostic factor both in progression-free survival and overall survival (p = 0.01, 0.01, respectively). ECOG Performance status was a significant prognostic factor in overall survival (p = 0.0009). In the combined analysis of stromal CD8+TIL and PD-L1, PD-L1 positive with high stromal CD8+TIL subgroups had a better prognosis than PD-L1 negative with low stromal CD8+TIL subgroups in progression-free survival (p = 0.006). Although these results require a further investigation, PD-L1 and ECOG Performance status and the combination of stromal CD8+TIL and PD-L1 positivity have potential as useful prognostic markers in patients of virus unrelated head and neck squamous cell carcinoma treated with nivolumab.

Expression of PD-L1 in Lung Carcinoma and Its Correlation with Histopathological Grade, Stage, and Survival of Patients.

Objective PD-L1, a 40 kDa type 1 transmembrane protein, suppresses the adaptive arm of the immune system. The interaction of PD-1 with the ligand PD-L1 inhibits cytokine production and plays a role in the progression of lung cancer. The present study was performed to observe the expression of PD-L1 in lung carcinoma patients and its correlation with histopathological grade, stage, and survival of patients. Materials and Methods This prospective study included all new cases of lung carcinoma diagnosed on histopathological or cytopathological examination over a period of 1 year. PD-L1 immunoexpression was statistically analyzed and graded according to the Tumor Proportion Score in all cases and correlated with histopathological grade, stage, and survival of patients. Results This study included 56 cases of lung carcinoma with 64.2% cases showing PD-L1 positivity, out of which 44.6% were non-small cell and 19.6% were small cell lung carcinoma. In all, 32.1% cases with lymphovascular invasion, 53.5% with necrosis, and 37.5% cases with greater than 5/10 HPF mitotic figures showed positive PD-L1 expression. Paired cell blocks and histopathology showed 70% concordance for PD-L1 expression. 16.1% cT3N1M0 cases and 25% stage IIIA cases showed PD-L1 positivity. In all, 60.7% patients with positive PD-L1 expression did not survive for 12 months following diagnosis. Conclusion PD-L1 immunoexpression was increased in lung carcinoma cases and was associated with poor histomorphological features including lymphovascular invasion, necrosis, and increased mitotic activity. PD-L1 correlated with cases having decreased 12-month survival and stage IIIA carcinoma. Thus, it may be useful in the stratification of patients who benefit from the PD-L1 targeted therapy.

Prevalence and prognostic significance of PD-L1, TIM-3 and B7-H3 expression in endometrial serous carcinoma

Endometrial serous carcinoma (ESC) is an aggressive type of endometrial carcinoma with a poor prognosis. Immune checkpoint blockade has evolved as a novel treatment option for endometrial cancers; however, data on expression of immune checkpoints that may be potential targets for immunotherapy in ESC are limited. We analyzed the prevalence and prognostic significance of PD-L1, TIM-3 and B7-H3 immune checkpoints in 99 ESC and evaluated their correlation with CD8 + tumor infiltrating lymphocytes. Applying the tumor proportion score (TPS) with a cutoff of 1%, PD-L1, TIM-3 and B7-H3 expression was present in 17%, 10% and 93% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, PD-L1, TIM-3 and B7-H3 expression was present in 63%, 67% and 94% of cases, respectively. Expression of these markers was largely independent of one another. PD-L1 correlated with higher CD8 + T-cell density when evaluated by either TPS (p = 0.02) or CPS (p < 0.0001). TIM-3 correlated with CD8 + T-cell density when evaluated by CPS (p < 0.0001). PD-L1 positivity was associated with improved overall survival (p = 0.038) when applying CPS. No association between PD-L1 expression and survival was found using TPS, and there was no association between TIM-3 or B7-H3 positivity and survival by either TPS or CPS. Using TPS, PD-L1 correlated with a higher tumor stage but not with survival, whereas the converse was true when PD-L1 was evaluated by CPS, suggesting that PD-L1 expression in immune cells correlates with prognosis and is independent of tumor stage. In conclusion, PD-L1, TIM-3 and B7-H3 may be potential therapeutic targets in selected patients with ESC. Further investigation of their roles as predictive biomarkers is needed.

Correlation of PD-L1 immunohistochemical expression with microsatellite instability and p53 status in endometrial carcinoma.

Endometrial carcinoma (EC) is the most common gynaecological cancer worldwide. The Cancer Genome Atlas molecular grouping of a given case of EC could be assessed by POLE gene mutation, mismatch repair (MMR) 'to reflect microsatellite instability' and p53 status, which has proved to be of prognostic value. Programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) are playing a progressively important role in tumour immunology and cancer treatment. To investigate PD-L1 immunohistochemical expression in EC in relation to MMR and p53 status. Associations between marker expression and different histopathological parameters were also investigated. This retrospective study was performed on archival biopsies of 170 cases of EC using a tissue microarray model. Immunohistochemical staining was applied using antibodies against PD-L1, MLH1, MSH2 and p53. The percentages of positivity were as follows: PD-L1, 19.6%; MLH1, 79.5%; MSH2, 78.5%; and p53 mutant, 13.8%. There was significant correlation between MLH1 expression and MSH2 expression (p=0.008). Tumour grade was significantly correlated with stage (p=0.005) and p53 mutant expression (p=0.008). Combined PD-L1 positivity and MMR deficiency showed significant correlation with the presence of lymphovascular space invasion (p=0.014). MSH2 negativity was significantly associated with poorer overall survival (p=0.014). A panel of immunohistochemical markers (PD-L1, MLH1, MSH2 and p53) could help to predict the prognosis and plan the treatment of patients with EC. MMR deficiency seems to be a good predictor for PD-L1 status, and therefore the response to potential PD-1/PD-L1 inhibitor therapy.

Efficacy and Safety of the PD-1 Inhibitor Combined with Albumin-Bound Paclitaxel and Nedaplatin in Preoperative Neoadjuvant Therapy of Unresectable Stage III Lung Squamous Cell Carcinoma.

To investigate the efficacy and safety of preoperative neoadjuvant therapy (PD-1 inhibitor plus nab-PTX and nedaplatin) for resectable stage III lung squamous cell carcinoma (SCC) patients. Patients with locally advanced lung SCC (stage IIIA, IIIB) who received PD-1 inhibitor combined with nab-PTX and NED between February 2019 and June 2021 in Weihai Municipal Hospital were included and underwent surgical treatment 4 weeks after 2-4 cycles neoadjuvant therapy. The rate of resection R0, the effective rate, the complete pathological remission rate (pCR) and the rate of major pathological remission (MPR) were observed. A total of 14 initially unresectable male patients with lung SCC were included and received neoadjuvant treatment after evaluation. Nine out of 14 patients (64.3%) experienced treatment-related adverse events (TRAE), among which 8 (57.1%) experienced grade (G) I-II TRAEs including nausea, vomiting, fatigue, constipation, elevated ALT and AST, hyperthyroidism, hypothyroidism, rash, granulocytopenia, and thrombocytopenia, and 1 (7.1%) experienced grade III-V TRAEs (G), including granulocytopenia and atelectasis. Thirteen patients (92.86%) achieved RECIST-assessed partial remission (PR), while 1 patient (7.14%) achieved stable disease (SD) on imaging assessment after neoadjuvant treatment and continued to be progression-free for 26 months. Of the 11 patients who underwent resection, all were alive and recurrence/progression-free. MPR and pCR were observed in 2 (18.18%) and 9 (81.82%), respectively. IHC results exhibited that all NSCLC patients exhibited positive PD-L1 expression (9/14, TPS ≥50% or greater; 5/14, 1% < TPS < 50%). Two were negative for ALK, EGFR, and ros-1, and the rest were not examined for driver oncogene mutation. The neoadjuvant therapy of the PD-1 inhibitor combined with nab-PTX and NED demonstrated remarkable therapeutic efficacy and good safety on stage III lung SCC without increasing the risk of TRAE, mortality and surgery-related complications, or impede surgery feasibility.

A potential treatment option for transformed small-cell lung cancer on PD-L1 inhibitor-based combination therapy improved survival

ObjectivesTransformed small-cell lung cancer (T-SCLC) has an extremely poor prognosis, and no remedies based on immunotherapy have been evaluated among T-SCLC patients. We retrospectively analysed the efficacy and safety of combining atezolizumab with chemotherapy for T-SCLC. MethodsForty-seven patients harbouring EGFR mutations who developed T-SCLC were enrolled. Eleven patients who used immunotherapy were defined as the I/O group, and the remaining 36 were defined as the Non-I/O group. Clinical characteristics, pathological data, and survival outcomes were collected. RNA sequencing and whole-exome sequencing (WES) were performed for in-depth analysis. ResultsAll patients received at least one line of EGFR-TKI before rebiopsy to confirm T-SCLC. Nine patients received atezolizumab-bevacizumab-carboplatin-paclitaxel (albumin-bound) (ABCP), and the remaining 2 received atezolizumab-etoposide-carboplatin (ECT) in the I/O group. The objective response rate was 73 % (8/11). The median progression-free survival (mPFS) of T-SCLC on post-transformation therapy with I/O group and Non-I/O group was 5.1 m and 4.1 m, respectively. The median post-T-SCLC overall survival of the I/O group was significantly longer than that Non-I/O group (20.2 m vs 7.9 m, P ＜ 0.01). T-SCLC harbouring EGFR L858R tended to be longer than EGFR 19del (mPFS: not reached vs 3.7 m, P = 0.11). Positive PD-L1 status was also associated with PFS benefits (mPFS: 6.0 m vs 3.7 m, P = 0.20). Furthermore, RNA sequencing revealed that expression of SFTPA1 is significantly higher in the durable clinical benefit group. WES showed that STC2 mutation is more frequently observed at the time-point immunotherapy acquired resistance. Combination therapy based on a PD-L1 inhibitor was well tolerated, and the safety profile was consistent with previously reported studies. ConclusionOur study first demonstrated that a PD-L1 inhibitor combined with chemotherapy ± bevacizumab could be a potential safe option for specific SCLC-transformed patients. Subsequent studies with more patients are essential to verify the efficacy and potential biomarkers.

Intratumor heterogeneity is associated with less CD8+ T cell infiltration and worse survival in patients with small cell lung cancer.

Small cell lung cancer (SCLC) is a heterogeneous malignancy with genetic and phenotypic disparity. However, the association between intratumor heterogeneity (ITH) and immunological features as well as the impact of ITH on prognosis has never been explored in SCLC. Hence, we investigated the relationship between ITH and their immunological features and explored the effect of ITH on overall survival (OS) in patients with SCLC. Programmed cell death-ligand 1 (PD-L1), CD8+ cell infiltration was calculated through immunohistochemical staining and tumor mutational burden (TMB), tumor neoantigen burden (TNB), and ITH levels via whole-exome sequencing (WES). Significant correlation was not found in ITH versus TMB, ITH versus TNB (P = 0.1821, P = 0.0612). No significant variation in ITH was found between negative PD-L1 SCLC patients and positive PD-L1 ones (P = 0.0610 for TPS, P = 0.6347 for CPS). Interestingly, we demonstrated the negative correlation between CD8+ T cell infiltration and ITH (P = 0.0220). More importantly, significant OS benefit was detected in ITH-low SCLC patients in comparison with ITH-high ones (P = 0.0049). ITH was an independent prognostic factor on OS with clinicopathological variables adjusted (HR, 2.044; 95% CI 1.190-3.512; P = 0.010). We also demonstrated significantly different driver genes and CNV between ITH-low and ITH-high SCLC. Our work pointed the negative association of ITH with CD8+ T cell infiltration and suggested ITH as a potential predictor of OS in SCLC, putting forward a direction for more precise and individualized therapeutic strategies for SCLC.

Clinical and prognostic implications of CD47 and PD-L1 expression in surgically resected small-cell lung cancer

Pharmacological inhibition of the immune-checkpoint molecule CD47 has shown promising results in preclinical small-cell lung cancer (SCLC) models, whereas anti-programmed death-ligand 1 (PD-L1) inhibitors have been recently implemented in the standard of care of advanced-stage SCLC patients. Nevertheless, the expression pattern, clinical relevance and prognostic implication of both CD47 and PD-L1 are rather controversial in surgically treated SCLC patients. In total, 104 Caucasian SCLC patients from two Central European thoracic centers were included in this study. CD47 and PD-L1 expression as well as the expression of the four major SCLC molecular subtype markers (ASCL1, NEUROD1, YAP1 and POU2F3) were measured by immunohistochemistry. Expression levels were independently evaluated and statistically correlated with clinicopathological data and survival. Positive CD47 and PD-L1 expressions were seen in 84.6% and 9.6% of the samples, respectively. Meanwhile, the tumor-associated stroma was positive for PD-L1 in 59.6% of the cases. Stromal PD-L1 expression correlated with longer overall survival (OS) (versus PD-L1-negative stroma; median OS was 42 versus 14 months, respectively, P= 0.003) and was confirmed as an independent predictor of favorable outcome upon multivariate analysis (hazard ratio 0.530, 95% confidence interval 0.298-0.943, P= 0.031). Notably, neither CD47 nor PD-L1 presence was related to a distinct molecular SCLC subtype. CD47 shows a remarkably high expression while tumoral PD-L1 expression is generally low in surgically treated SCLC. Importantly, stromal PD-L1 expression may indicate a favorable clinical outcome and serve as a novel prognostic factor in these patients. Additional studies are warranted to further investigate the clinical impact of CD47 and PD-L1 expression in SCLC.

Oncogenic JAK2/STAT Signaling Molecules Activation Is Associated with PD-L1 Upregulation in Acute Myeloid Leukemia

Background: Increased evidence suggests the PD-1/PD-L1 axis is involved in the oncogenesis of myeloid neoplasms through promoting cancer immune escape, which suggests potential benefits by blockade of this axis in these neoplasms by immune checkpoint inhibitors. However, the ongoing clinical trials on PD-1/PD-L1 blocker in MDS/AML have reported controversial results, unlike the promising therapeutic efficacy shown in solid tumors. Identifying reliable biomarkers and scoring of PD-L1 expression and targeting the subgroup of patients who might benefit the most from PD-1/PD-L1 blockade are promising directions. Janus kinase 2 (JAK2) pathway activation has been shown to enhance PD-L1 expression in malignancies by in vitro studies. Here, we investigated PD-L1 expression associated with JAK2/STAT oncogenic pathway in AML patients using the combined positive score (CPS) algorithm. Methods: Immunohistochemical staining (IHC) for PD-L1 and pSTAT3 was performed on the marrow biopsies of 31 AML patients (39 cases), including 11 patients (17 cases) with JAK2/STAT mutation and 20 patients (22 cases) with wild-type JAK2/STAT. For PD-L1 IHC, the cases were evaluated for variable staining intensity (weak, moderate, strong) and membrane staining pattern (partial, complete) in tumor and mononuclear immune cells. The PD-L1 CPS was calculated by summing the number of all PD-L1-stained cells and dividing by the total number of tumor cells, multiplied by 100. The pSTAT3 was evaluated by percentage of positive tumor cells among the total tumor cells. The status of JAK2 / STAT mutations and other molecular abnormalities was evaluated by next generation sequencing (NGS) study. Results: Among patients of JAK2/STAT mutations, nine with JAK2 V617F exon 14 mutations, one with JAK2 I540-E543delinsRG exon 12 mutation and one with STAT5B N642H mutation. Evaluation of PD-L1 expression by IHC revealed four major cell staining patterns in our study cohort: 1. Negative (Figure 1A); 2. Partial weak membrane staining (Figure 1B, CPS<10); 3. Partial-complete, weak-moderate membrane staining (Figure 1C, CPS≥10); 4. Complete, moderate-strong membrane staining (Figure 1D, CPS≥10). Patterns 3-4 were classified as positive PD-L1 staining with a CPS cut-off of 10 (CPS≥10). Comparison of the PD-L1 CPS score in the JAK2/STAT wild-type versus the JAK2/STAT mutant groups revealed significantly higher PD-L1 CPS score in the JAK2/STAT mutant group (Figure 1K, P<0.0001, CPS≥10). One patient showed wild-type JAK2 at initial AML diagnosis. At two subsequent relapses, gain of JAK2 exon 12 (I540-E543delinsRG) mutation with increased VAF from 17% to 41% was detected. Interestingly, PD-L1 expression was correspondingly increased with CPS from 25 to 75 in these subsequent biopsies (Figure 1E, F). In another case with wild-type JAK2, STAT5B N642H mutation was detected and strong PD-L1 expression with CPS of 80 was obtained (Figure 1G). To address if JAK2/STAT3 axis is involved in PD-L1 upregulation, we assessed STAT3 phosphorylation by IHC in JAK2 wild-type and mutated groups. The results revealed significantly increased p-STAT3 expression in the JAK2 mutant compared with the wild-type cases, supporting the idea that oncogenic JAK2 mutation leads to STAT3 phosphorylation (Figure 1H-J). Furthermore, p-STAT3 expression showed a significant positive correlation with the PD-L1 CPS score (Figure 1H, r=0.8663, P<0.0001). Conclusion: In the AML patients with oncogenic JAK2 or its downstream signaling molecule STAT5B mutations, there is significantly enhanced PD-L1 expression. In addition, we proposed that the PD-L1 CPS positivity cut-off score of 10 being used as a quantitative measure of PD-L1 expression in this heterogeneous myeloid neoplasm. In the patient with relapsing AML, PD-L1 expression showed a positive correlation with increased JAK2 mutant VAF. Independent of the oncogenic JAK2 mutation, PD-L1 expression upregulation was also noted with STAT5B N642H mutation. Lastly, we demonstrated enhanced p-STAT3 expression in JAK2 mutant cases and a positive correlation between p-STAT3 and PD-L1 expression. Taken together, these data support the model that JAK2/STAT pathway activation is associated with PD-L1 upregulation in AML patients (Figure 1I), together with the proposed PD-L1 CPS cut-off of 10, can serve as scanning biomarkers to select relevant subgroups of AML patients for PD-1/PD-L1 immunotherapeutic study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal